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Systematic Bacteriology

for Pharmacy Students

Haemophilus, Bordetella, Brucella, & Francisella

Instructor: Mohsen Amin

The Haemophilus spp.

• Small, Gram-negative, pleomorphic, require

enriched media containing blood or its

derivatives

• Haemophilus influenzae type b causes

meningitis in children

• Haemophilus ducreyi, a sexually transmitted

pathogen, causes chancroid

Haemophilus influenzae

• Found on the upper respiratory tract

• Important cause of meningitis in children

• Morphology: short 1.5 µm coccoid bacilli

• Culture: Flat grayish brown colonies on

chocolate agar + IsoVitaleX

• Satellite phenomenon on on sheep blood

agar

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IsoVitaleXTM

composition

Haemophilus influenzae

Satellite Test

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• Growth characteristics: Identification is

based on the need for certain growth factors

called X and V

– Factor X acts as hemin

– Factor V can be replaced by NAD

– Staphylococci on blood agar release NAD

• Variation: pleomorphic; loss of capsule

• Transformation: transmissible plasmids

(ampicillin and chloramphenicol resistance)

Haemophilus influenzae

Hemin (trade name Panhematin)

• Hematin (factor X) has a hydroxide ligand in place of the

chloride

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H. influenzae on chocolate agar

Antigenic structure

• Capsular polysaccharides of one of six

types (a-f). Type b: polyribose-ribitol

phosphate (PRP) is a major virulence

factor

• LPS is analogous to neisserial LPS

• Outer membrane proteins

Antigenic typing

• Encapsulated H. influenzae can be typed

by slide agglutination, coagglutination with

staphylococci, or agglutination of latex

particles coated with type-specific Ab

• Capsule swelling test with specific

antiserum is analogous to the quelung test

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Pathogenesis

• Capsule is antiphagocytic

• The nonencapsulated H. Influenzae is a

member of normal flora (50-80%)

• Meningitis, pneumonia, empyema,

epiglottitis, cellulitis, septic arthritis

• H. influenzae type b and pneumococci are

two of the most common etiologic agents

of bacterial otitis media and acute sinusitis

Diagnostic tests

• Specimens: nasopharyngeal swabs, pus,

blood, and spinal fluid for smears

• Direct identification: immunologic

detection of antigens in spinal fluid. Gram

stain

• Culture: IsoVitaleX-enriched chocolate

agar

Gram-negative H. Influenzae in sputum

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Immunity

• Infants may have serum Ab from the mother

• By age 3-5 years, many unimmunized

children have naturally acquired anti-PRP Ab

• Immunization of children with H. influenzae

type b conjugate vaccine induces the same

Ab

Treatment

• Ampicillin and cefotaxime

• Up to 25% of strains produce β-lactamase

under control of a transmissible plasmid and

are resistant

Epidemiology and control

• Person to person transmission

• Haemophilus b conjugate vaccine:

– PRPHbOC

– PRP-OMPC

– PRP-T

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Haemophilus ducreyi

• Causative agent of chancroid (soft chancre)

• Ragged ulcers with marked swelling and tenderness. Gram-negative rods occur in strands

• Requires X factor but not V factor. Grows on chocolate agar + 1% IsoVitaleX

• There is no permanent immunity following chancroid infection

• Treatment: ceftriaxone, TMP-SXT, erythromycin

Other Haemophilus spp.

• H. haemolyticus is markedly hemolytic;

rare upper respiratory tract infections in

childhood

• H. parainfluenzae is the normal inhabitant

of the respiratory tract; occasionally

involved in endocarditis and urethritis

The Bordetellae

• Bordetella pertussis causes whooping cough (pertussis)

• Boredetella parapertussis can cause a similar disease

• Bordetella bronchiseptica causes diseases in animals such as kennel cough in dogs and snuffles in rabbits, and only occasionally in humans

• Bordetella avium causes turkey coryza

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Bordetella pertussis

• Morphology: minute Gram-negative

coccobacilli. Bipolar metachromatic

granules (with toluidine blue); capsule

• Culture: Bordet-Gengou medium (potato-

blood-glycerol agar) + penicillin G;

charcoal-containing medium can be used

• Virulent type is hemolytic and toxin-

producing

Antigenic structure of B. pertussis

• Filamentous hemagglutinin and pili

• Pertussis toxin: promotes lymphocytosis, sensitization to histamine, and enhanced insulin secretion and has ADP-ribosylating activity

• Adenylate cyclase toxin

• Dermonecrotic toxin

• Hemolysin

• Tracheal cytotoxin

• LPS

• Pertactin OMP

Lymphocytosis in whooping cough

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Clinical findings

• Incubation period: about 2 weeks

• Catarrhal stage: mild coughing and

sneezing. The patient is highly infectious

• Paroxysmal stage: cough develops its

characteristic “whoop”. Exhaustion and

vomiting, cyanosis, and convulsion

• Blood is not invaded

Diagnostic tests

• Specimens: nasal wash, nasopharyngeal

swabs, cough plate

• Direct fluorescent Ab test

• Culture: Bordet-Gengou or charcoal-

containing medium

• PCR

• Serology

Immunity

• The first defense is the Ab that prevents

attachment

• Reinfections occur years later in adults

may be severe

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Treatment

• Erythromycin during the catarrhal stage

• Treatment after onset of the paroxysmal

phase rarely alters the clinical course

Prevention

• 5 injections of pertussis vaccine are

recommended prior to school entry

• Acellular pertussis vaccine: inactivated

pertussis toxin, filamentous hemagglutinin,

fimbrial proteins, and pertactin

• Usually administered in combination with

toxoid of diphtheria and tetanus (DTaP)

• Prophylactic administration of erythromycin

Epidemiology and control

• Whooping cough is endemic in most

densely populated areas

• The source of infection is a patient in the

early catarrhal stage

• Most cases occur in children under age 5

years

• Control rests mainly on adequate active

immunization of all infants

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The Brucellae

• Obligate parasites of animals and humans

• Relatively inactive metabolically

• The disease in humans, brucellosis

(undulant fever, Malta fever), is

characterized by an acute bacteremic

phase followed by a chronic stage

The Brucellae

Species Host

Brucella melitensis Goats

Brucella suis Swine

Brucella abortus Cattle

Brucella canis Dogs

The Brucellae

• Morphology: Gram-negative coccobacilli

• Culture: virulent colonies are smooth,

transparent on enriched media. Fresh

specimens from animal/human sources

inoculated on trypticase-soy agar or blood

culture media

• Catalase and oxidase positive

• Killed in milk by pasteurization

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Pathogenesis

• The common portal of entry: intestinal

tract, mucous membranes, and skin

• Cheese made from unpasteurized goat’s

milk

• The bacteria enter lymphatic channels,

regional lymph nodes and the bloodstream

• Granulomatous nodules in lymphatic

tissue, liver, spleen, and bone marrow

Pathogenic differences

• B. abortus and B. canis cause mild disease without suppurative complications; noncaseating granulomas

• B. suis causes chronic disease with suppurative lesions; caseating granulomas

• B. melitensis infection is more acute and severe

• There is no erythritol in human placentas, and abortion is not part of brucellosis in humans

Clinical findings

• Incubation period: 1-6 weeks

• Malaise, fever, weakness, aches, and

sweats

• Gastrointestinal and nervous symptoms

• Lymph node enlargement

• Hepatitis accompanied by jaundice

• Osteomyelitis

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Diagnostic tests

• Specimens: Blood, biopsy materials

• Culture: Brucella agar, trypticase soy agar

with or without 5% sheep blood, brain

heart infusion medium, and chocolate

agar. Liquid medium used to culture

Mycobacterium tuberculosis also supports

the growth of some strains

Diagnostic tests

• A positive urease test is characteristic of

Brucella spp.

• Agglutination test: IgG agglutinin titers

above 1:80

Treatment

• Tetracycline and ampicillin

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Epidemiology and control

• Humans infected in one of three ways:

– Eating undercooked meat or consuming unpasteurized/raw dairy products

– Inhalation

– Through skin, wounds or mucous membranes

• Active immunization of humans is experimental

• Control rests on limitation of spread and pasteurization

Francisella tularensis & tularemia

• Highly infectious (category A select agent).

Type A is the most virulent

• Mortality rate: 30-60% of the cases

• Widely found in animal reservoirs and

aquatic environments

• Four recognized subspecies of Francisella

tularensis: tularensis, holarctica,

mediasiatica, and novicida

Morphology & Identification

• Small Gram-negative coccobacilli

• Culture: Chocolate agar (contains hemin), modified Thayer-Martin agar, and buffered charcoal yeast extract (BCYE) agar

• Serology: All isolates are serologically identical (polysaccharide Ag and protein Ag)

• There are two major biotypes, A and B

• Biotype A: ferments glycerol and contains citrulline ureidase

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Pathogenesis

• Entry route: skin, mucous membranes, or

inhalation of 50 organisms

• Inflammatory, ulcerating papule develops

• Regional lymph nodes enlarge and may

become necrotic

• Localized pneumonitis (pneumonic tularemia)

• Oculoglandular tularemia

• Fever, malaise, headache and pain

Oculoglandular tularemia

Treatment

• Streptomycin or gentamicin for 10 days

• Tetracycline may be equally effective

• Resistant to all β-lactam antibiotics

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Epidemiology and control

Review questions

• Which of the following subspecies of

Francisella tularensis is the most virulent

for humans?

A. tularensis

B. holarctica

C. mediasiatica

D. novicida

• All of the following statements regarding the

etiologic agent of chancroid are correct except:

A. The organism is a small Gram-negative rod

B. The organism requires X factor but not V factor

C. The organism grows well on standard chocolate

agar

D. On Gram stain of lesions the organism occurs

in strands

E. The organism is susceptible to erythromycin

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• All the following regarding acellular pertussis vaccines are correct except:

A. All formulations of the vaccine contain at least two antigens

B. The vaccine has replaced the whole cell vaccine in the childhood vaccine series

C. All children should receive five doses

D. The vaccine is approved only for young children and adolescents

E. The vaccine is safer than and as immunogenic as whole cell vaccines